Eric J. Ackerman

Aberrant mobility phenomena of the DNA repair protein XPA

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time‐resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI‐FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post‐translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS‐PAGE (multiple bands from ∼40–45 kDa) and gel filtration chromatography (∼92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS‐PAGE yielded a single 42‐kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellmans reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a ∼32‐kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPAs anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA ± cisplatin ± mismatches. xXPA showed at least 10‐fold higher affinity for cisplatin DNA ± mismatches compared to undamaged DNA ± mismatches. In all cases, DNA binding did not correct xXPAs anomalous migration. To test predictions that a Glu‐rich region (EEEEAEE) and/or disordered N‐ and C‐terminal domains were responsible for xXPAs aberrant mobility, the molecular weights of partial proteolytic fragments from ∼5 to 25 kDa separated by reverse‐phase HPLC and precisely determined by ESI‐FTICR MS were correlated with their migration on SDS‐PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%–50% larger molecular weights than expected. Thus, both the disordered domains and the Glu‐rich region in xXPA are primarily responsible for the aberrant mobility phenomena.

Identification of intrinsic order and disorder in the DNA repair protein XPA

The DNA‐repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full‐length protein, the minimal DNA‐binding domain, revealed that one‐third of this molecule was disordered. To better characterize structural features of full‐length XPA, we performed time‐resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS‐PAGE. The molecular weight of the full‐length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS‐PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N‐ and C‐terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well‐characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85‐I179) despite 14 possible cut sites. For the full‐length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends.

Abstract Semenenko, V. A., Stewart, R. D. and Ackerman, E. J. Monte Carlo Simulation of Base and Nucleotide Excision Repair of Clustered DNA Damage Sites. I. Model Properties and Predicted Trends. Radiat. Res. 164, 180–193 (2005). DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

Clay Nanoparticle-Supported Single-Molecule Fluorescence Spectroelectrochemistry

Here we report that clay nanoparticles allow formation of a modified transparent electrode, spontaneous adsorption of fluorescent redox molecules on the clay layer, and thus the subsequent observation of single-molecule fluorescence spectroelectrochemistry. We can trace single-molecule fluorescence spectroelectrochemistry by probing the fluorescence intensity change of individually immobilized single redox molecules modulated via cyclic voltammetric potential scanning. This work opens a new approach to explore interfacial electron transfer mechanisms of redox reactions.

A combined confocal and magnetic resonance microscope for biological studies

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

Tight correlation between inhibition of DNA repair in vitro and transcription factor IIIA binding in a 5S ribosomal RNA gene

UV‐induced photoproducts (cyclobutane pyrimidine dimers, CPDs) in DNA are removed by nucleotide excision repair (NER), and the presence of transcription factors on DNA can restrict the accessibility of NER enzymes. We have investigatigated the modulation of NER in a gene promoter using the Xenopus transcription factor IIIA (TFIIIA)–5S rDNA complex and Xenopus oocyte nuclear extracts. TFIIIA alters CPD formation primarily in the transcribed strand of the 50 bp internal control region (ICR) of 5S rDNA. During NER in vitro, CPD removal is reduced at most sites in both strands of the ICR when TFIIIA is bound. Efficient repair occurs just outside the TFIIIA‐binding site (within 10 bp), and in the absence of 5S rRNA transcription. Interestingly, three CPD sites within the ICR [+56, +75 (transcribed strand) and +73 (non‐transcribed strand)] are repaired rapidly when TFIIIA is bound, while CPDs within ∼5 bases of these sites are repaired much more slowly. CPDs at these three sites may partially displace TFIIIA, thereby enabling rapid repair. However, TFIIIA is not completely displaced during NER, at least at sites outside the ICR, even though the NER complex could be sterically hindered by TFIIIA. Such inefficient repair of transcription factor binding sites could increase mutation frequency in regulatory regions of genes.

Combined confocal and magnetic resonance microscopy

Confocal fluorescence optical microscopy and magnetic resonance microscopy are each used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each microscope individually. In this paper two configurations of a combined confocal and magnetic resonance microscope are described. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas the second configuration is designed for the investigation of monolayers of mammalian cells. In both cases the sample compartment is part of a temperature regulated perfusion system. Images obtained with the combined system are shown forXenopus laevis oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.

Nucleotide Excision Repair in Nuclear Extracts from Xenopus Oocytes

Limited nucleotide excision repair (NER) requires at least {approx}40 proteins in extracts from purified proteins, although perhaps hundreds of proteins may influence DNA repair in cells. For efficient DNA repair in extracts, it is important to utilize a system containing large quantities of active DNA repair proteins uncontaminated with nonspecific nucleases. Unlike extracts from mammalian cells that repair {approx}2% of the input DNA, both injected Xenopus oocytes and oocyte nuclear extracts can repair {approx}100% of the input damaged DNA by NER with little or no synthesis on undamaged control substrate. Repair activity in extracts can be inactivated with antibodies and/or inhibitors, and then repair can be restored by addition of exogenous proteins. A further advantage of the Xenopus system is that results obtained from injection experiments in living cells can be compared to results obtained in nuclear extracts.

Single-molecule electron transfer reactions in nanomaterials

Here we report the study of single molecule electron transfer dynamics by coupling fluorescence microscopy at a conventional electrochemical cell. The single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution and on nanoparticle surface were studied. We observed that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning. We attribute the fluorescence intensity change of single cresyl violet molecules to the electron transfer reaction driven by the electrochemical potential.

An integrated confocal and magnetic resonance microscope for observing living cells

We have combined a confocal fluorescence optical microscope and a magnetic resonance microscope into one instrument to image simultaneously a living cell system. The high spatial resolution confocal and high spectral resolution magnetic resonance microscope produces a set of closely linked, complementary images. To enhance the value of this image set, we have also developed a statistical method, local linear modeling, to predict a high spatial resolution magnetic resonance image from the confocal optical and magnetic resonance image pair. This high spatial, high spectral resolution image offers insights not available in images obtained from separate studies, nor available in the viewing the image pair as is. This paper presents first results from application of this instrument and local linear modeling, a statistical prediction technique, to a calibration phantom and a Xenopus laevis oocyte cluster.